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首页> 外文期刊>American Journal of Physiology >Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages.
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Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages.

机译：RAW 264.7巨噬细胞中TNF生物合成与CaM依赖型诱导型一氧化氮合酶的活化之间的功能联系。

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Inflammatory responses stimulated by bacterial endotoxin LPS involve Ca2+-mediated signaling, yet the cellular sensors that determine cell fate in response to LPS remain poorly understood. We report that exposure of RAW 264.7 macrophage-like cells to LPS induces a rapid increase in CaM abundance, which is associated with the modulation of the inflammatory response. Increases in CaM abundance precede nuclear localization of key transcription factors (i.e., NF-kappaB p65 subunit, phospho-c-Jun, Sp1) and subsequent increases in the proinflammatory cytokine TNF-alpha and inducible nitric oxide synthase (iNOS). Cellular apoptosis after LPS challenge is blocked upon inhibition of iNOS activity using the pharmacological inhibitor 1400W. LPS-mediated iNOS expression and apoptosis also were inhibited by siRNA-mediated silencing of TNF induction, indicating TNF induction both precedes and is necessary for subsequent regulation of iNOS expression. Increasing the level of cellular CaM by stable transfection results in reductions in LPS-induced expression of TNF and iNOS, along with reduced activation of their transcriptional regulators and concomitant protection against apoptosis. Thus the level of CaM available for Ca2+-dependent signaling regulation plays a key role in determining the expression of the proinflammatory and proapoptotic cascade during cellular activation by LPS. These results indicate a previously unrecognized central role for CaM in maintaining cellular homeostasis in response to LPS such that, under resting conditions, cellular concentrations of CaM are sufficient to inhibit the biosynthesis of proinflammatory mediators associated with macrophage activation. Although CaM and iNOS protein levels are coordinately increased as part of the oxidative burst, limiting cellular concentrations of CaM due to association with iNOS (and other high-affinity binders) commit the cell to an unchecked inflammatory cascade leading to apoptosis.
机译：细菌内毒素LPS刺激的炎症反应涉及Ca2 +介导的信号传导，但对LPS反应确定细胞命运的细胞传感器了解甚少。我们报告说，RAW 264.7巨噬细胞样细胞暴露于LPS会引起CaM丰度的快速增加，这与炎症反应的调节有关。 CaM丰度增加先于关键转录因子（即NF-κBp65亚基，磷酸化c-Jun，Sp1）的核定位，然后是促炎性细胞因子TNF-α和诱导型一氧化氮合酶（iNOS）的增加。使用药理抑制剂1400W抑制iNOS活性后，LPS攻击后的细胞凋亡被阻断。 LPS介导的iNOS表达和凋亡也受到siRNA介导的TNF诱导沉默的抑制，这表明TNF诱导既在iNOS表达之前，也是后续调节iNOS表达所必需的。通过稳定的转染增加细胞CaM的水平会导致LPS诱导的TNF和iNOS的表达减少，同时其转录调节因子的激活减少，并同时具有抗凋亡的保护作用。因此，可用于Ca 2+依赖性信号传导调节的CaM水平在确定LPS激活细胞过程中促炎和促凋亡级联的表达中起关键作用。这些结果表明，CaM在维持对LPS的反应中保持细胞稳态方面的作用尚未得到认可，因此在静止条件下，CaM的细胞浓度足以抑制与巨噬细胞活化有关的促炎性介质的生物合成。尽管CaM和iNOS蛋白水平作为氧化性爆发的一部分而协同增加，但由于与iNOS（和其他高亲和力结合剂）缔合而导致的有限的CaM细胞浓度将使细胞发生不受控制的炎症级联反应，从而导致细胞凋亡。

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